Regeneration and genetic transformation of acacia mangium

ABSTRACT

The present invention is directed to a method of  Acacia mangium  regeneration through organogenesis and a method of genetic transformation of  Acacia mangium.  The method of regeneration comprises inducing callus from different parts of seedlings and vegetatively micropropagated plantlets as explants; adventitious bud induction followed by pinnate leaf and bud elongation and eventually, elongated shoots were induced to root. Based on the regeneration system, a marker gene GUS under cauliflower mosaic virus promoter was introduced to  Acacia mangium  via  Agrobacterium  infection. GUS staining in the regenerated plants and Southern blot hybridization prove the incorporation of the foreign gene into the host genome and expression of the foreign gene.

This application is a division of application Ser. No. 09/936,612, filedJan. 2, 2002.

BACKGROUND OF THE INVENTION

Forests are very important to the world economy and for maintaining andpreserving our ecosystem. Forest trees have a wide range of commercialuses (timber for construction, raw material for paper and pulpproduction, and as an energy source). The global demand for woodproducts (mostly for paper and pulp and firewood in developingcountries) has been increasing year by year when the natural forests arein short supply. Re-forestation is the solution to meeting suchincreasing demand. Usually, the fast-growing, widely adapted treespecies are chosen for re-forestation. Most tree-improvement programsare based on the management of genetic resources, including theselection of superior clones from existing forests, the conservation ofgenetic variability, partially controlled propagation and classicalbreeding for desired traits. In spite of the fact that it usually takesseveral generations to breed, this traditional breeding has beensuccessful in getting elite trees with fast and uniform growth. However,many other traits such as disease and insect resistance, differentlignin composition and content are difficult to acquire mainly due tohigh heterozygosity in tree species and big segregation population.Moreover, the gene(s) conferring certain phenotypes like diseaseresistance may not be in the gene pool at all. On the other hand,molecular breeding based on genetic transformation of tree speciesoffers the possibility to introduce a particular phenotype withoutaffecting the genetic background of a cultivar. Genetic transformationin Populus species and Eucalyptus species enabled some success inmodification of lignin content (Tzfira et al., 1998; Robinson, 1999).The precondition of molecular breeding of forest tree species is theavailability of a reliable and reproducible genetic transformationmethod, which in turn relies on a system of regeneration of one wholeplant from a single cell.

Genus Acacia comprises about 1200 tropical and subtropical tree species.It belongs to the family Mimosaceae. Acacia mangium is a multipurpose,fast growing and nitrogen fixing elite tropical legume tree. An adulttree is up to 30-metres tall and its bole (trunk) is often straight toover half the total height. The true leaf of a seedling is a fern-likepinnate leaf. The first pinnate leaf of a seedling has 6 or 8 leaflets,and then the bipinnate leaf develops from the second pinnate leaf on aseedling. Usually when the young seedling grows up to 8-12 bipinnateleaves, the petiole is dilated into a phyllode, while the leaflets abortcompletely and the true leaf disappears from the young seedling.Phyllodes are flattened leaf stems which look and act like the ordinaryleaves of other plants. Branchlets, phyllodes and petioles are glabrousor slightly scurfy. Phyllodes are 5-10 cm broad, 2-4 times as long asbroad, dark green, and are chartaceous when dry. The phyllodes have 3-4longitudinal main nerves which join on the dorsal margin at the base ofthe phyllode. Secondary nerves are fine and inconspicuous.

Flowers are in loose spikes to 10 cm long and are solitary or paired inthe upper axils. Flowers are pentamerous and the calyx is 0.6-0.8 mmlong with short obtuse lobes. The corolla is twice as long as the calyx.Pods are linear, glabrous, 3-5 mm broad, about 7.5 cm long when green,woody, coiled and brackish-brown when mature, and depressed between theseeds. Seeds are lustrous, black, ellipsoid, ovate or oblong, 3.5×2.5mm, with the orangish funicle forming a fleshy aril beneath the seed(Duke, 1984). Due to A. mangium fast growth, tolerance of infertile soiland high quality fibre, it has been increasingly used for reforestationplantation and soil rehabilitation in degraded soil. Its plantation hasbeen studied for many years in many countries or regions, especially intropical regions and subtropical regions, such as Australian, Indonesia,Malaysia, India, Thailand, Hawaii, China and Taiwan. Many A. mangiumplantations have been established in acidic soil or abandoned land orImperata grassland, for example, in Bangladesh (Latif et al., 1995); inSabah (Latif et al., 1995; Williams et al., 1992) and in Serdong (Majidet al., 1994; Awang, 1994) in Malaysia; Sangmelina, Cameroon, Kenya(Duguma et al., 1994); Skaerat, Thailand (Khemnark, 1994); Hawaii, USA(Cole et al., 1996); Bogor (Anwar, 1992; Wibowo et al., 1992), Paseh andKadipaten (Widiarti and Alrasjid, 1987) in Indonesia; Bengal, India(Basu et al., 1987), etc.

Indonesia, with some of the world's largest paper and pulp mills, hasbeen increasingly relying on plantations as the source of wood and A.mangium is the preferred choice. Asia Paper and Pulp group has twoaffiliate companies with a total concession of 540,000 hectares. By1996, one company had planted 123,000 hectares of A. mangium, about 90%of all its plantation, which represented 180 million seedlings. It isestimated that by 2004, Asia Paper and Pulp group will virtually sourceall its wood from plantation, mainly A. mangium plantation (Bayliss,1998a; Bayliss, 1998b).

Besides its use for paper and pulp, A. mangium timber can be used forother applications such as cement banded particle board, plywood anddecorative panel manufacture (Yusoff et al., 1989; Wong et al., 1988).

A large scale of mono-plantation of vegetative propagation from a singleplant faces a higher risk of disease infection. It has been found thatdiseases spread very fast in this kind of plantation and cause largeeconomic losses. Many diseases devastate A. mangium: Cinnamon fungus(Phytophehera cinnamomi) infection results in collapse and death ofplants; Seedling blight, defoliation and dieback due to Glomerellacingulata caused serious losses to A. mangium in nurseries. Leaf spot byCyclindrocladium quinqueseptatum causes defoliation of seedlings andyoung trees; Powdery mildew (Oidium spp) severely affected A. mangiumseedlings in nurseries in Thailand; Red rot diseases, caused byganoderma sp. affects A. mangium in Malaysia; Brown root disease causedby Phellinus noxium affects A. mangium in Malaysia and the Solomonislands (Simmons, 1987; Gutteridge and Shelton, 1994). Traditionalbreeding has been less successful in getting disease resistance in A.mangium, mostly likely due to lack of such a gene in the natural genepool. Molecular breeding by introducing foreign disease resistance genesbecomes an important option.

Studies on A. mangium tissue culture have been limited tomicropropagation (Bhaskan and Subbash, 1996; Ahmad, 1991; Galiana etal., 1991a; Galiana et al., 1991b). A combination of traditionalbreeding of elite trees and techniques of large-scale vegetativepropagation make the large-scale plantation possible. There has been noreport on regeneration or genetic transformation of A. mangium. Ourinvention describes the conditions for regeneration of whole plant viaorganogenesis and a genetic transformation system.

The publications and other materials used herein to illuminate thebackground of the invention or provide additional details respecting thepractice, are incorporated by reference, and for convenience arerespectively grouped in the appended List of References.

SUMMARY OF THE INVENTION

A. System of Regeneration from Callus in Acacia mangium

Different parts of Acacia mangium seedlings or vegetativemicropropagated plantlets (hypocotyl, cotyledon, leaflet, petiole andstem) were used as explants. The seedlings were from isolated seedembryo culture and the plantlets were obtained by micropropagation frommeristem regions of a 2-year old tree. Callus formation was induced byMS basic medium (Murashige and Skoog, 1962) supplemented with auxins(either 2,4-D from 0.5 mg/l to 5 mg/l or α-naphthaleneacetic acid (NAA)from 0.5 mg/l to 2 mg/l) and cytokinins (either kinetin (KT) 0.5 mg/l to3.0 mg/l or 6-benzylaminopurine (6-BA or BA) 0.5 mg/l to 3.0 mg/l).

Adventitious bud induction was achieved on medium AM-265 (MS basicmedium with thidiazuron (1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea or TDZ)1.0 mg/l, indole-3-acetic acid (IAA) 0.25 mg/l, casein enzymatichydrolysate (CH) 100 mg/l, L-ascorbic acid (vitamin C or Vc) 100 mg/l,L-glutamine (Gln) 150 mg/l, L-asparagine monohydrate (Asn) 150 mg/l,L-proline (Pro) 150 mg/l, pH 5.8 after autoclaving, phytagel 0.275 or0.30%, or agar 0.8%, sucrose 30 g/L) under the growth conditions of 16/8hours light/dark (L/D) cycle and at 28° C.

Pinnate leaf formation and adventitious bud elongation were obtained onmedium AM-337 (MS basic medium with TDZ 0.01 mg/l, CH 100 mg/l, Vc 100mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH 5.8 afterautoclaving, phytagel 0.275 or 0.30%, or agar 0.8%, sucrose 30 g/L), orAM-41 (MS basic medium supplemented with 6-BA 2 mg/l, CH 100 mg/l, Vc100 mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH 5.8 afterautoclaving, phytagel 0.275 or 0.30%, or agar 0.8%, sucrose 30 g/L)supplemented with gibberellic acid (GA₃) 0.5-2.5 mg/l. The growthconditions are 16/8 hrs (L/D) cycle and at 28° C.

Adventitious buds formed roots and complete plantlets were obtained onMS or Y₂MS basic medium supplemented with NAA 0.5 to 3.0 mg/l combinedwith KT 0-0.5 mg/l or IBA (indole butyric acid) 0.01-0.5 mg/l and CH 100mg/l, Vc 100 mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH 5.8after autoclaving, phytagel 0.35%, or agar 1.4%, sucrose 20 or 30 g/Lunder the growth conditions of 16/8 hrs (L/D) cycle and at 28° C.Complete plantlets were transplanted into peat soil: white sand (3:1) ina growth chamber at a photoperiod of 16/8 hrs (L/D) and 25° C.

B. System of Genetic Transformation of A. mangium

Based on the above established regeneration system, Agrobacterium strainLBA4404 (Ooms et al., 1981) harbouring binary vector pBI121 in which aGUS gene is under regulation of an 800 bp cauliflower mosaic viruspromoter was used for genetic transformation of A. mangium. TheAgrobacteria with the plasmid were co-cultured with the followingexplants: (1) Young adventitious shoots vegetatively micropropagatedfrom auxiliary buds dissected from a 2-year old tree. The shootsconsisted of young stem and pinnate leaves but no phyllodes. Stem piecesof about 2-3 cm in length after separating pinnate leaf and auxiliarybuds were used as explants. (2) Petiole pieces of 1-2 cm in length andleaflet pieces of 0.4-0.5×0.3-0.4 (cm) from seedlings derived fromembryo culture were used as explants. The explants were pre-cultured onAM-265 for three days at 16/8 hrs (L/D) cycle and at 28° C. beforeinfection with activated Agrobacterium for 15 minutes. Infected explantswere then cultured for 3 days in the same media in the dark at 22° C.They were then washed and put into AM-265 to induce callus andadventitious buds. Transgenic plants were selected with G418 in media infour stages: G418 12 mg/l for 25 days, G418 20 mg/l for 60 days, G418 30mg/l for 25 days and then on G418 12 mg/l. Adventitious bud elongationwas induced by AM-265 with GA₃ 2.5 to 5 mg/l in the presence of G418 12mg/l. For pinnate leaf formation, adventitious buds were transferredinto AM-41 with GA₃ 2.5 mg/l at growth conditions of 16/8 hours (L/D)cycle and 28° C. Regenerated shoots were excised and checked for GUSstaining and 16% tested positive. Putative transgenic adventitious budswere transferred into AM-357 or AM-451 with or without G418 (10 mg/l) atthe photoperiod of 16/8 hours (L/D) to induce rooting. Southern blothybridization showed positive results when NPTII gene was used as probe.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-E show adventitious bud induction from leaf. FIG. 1A showscallus induction. FIG. 1B shows adventitious bud recovery. FIGS. 1C, 1Dand 1E show plumule formation and adventitious bud elongation.

FIGS. 2A-C show Acacia mangium regeneration from petiole. FIG. 2C showscallus induction. FIG. 2B shows adventitious bud recovery. FIG. 2C showsplumule formation and adventitious bud elongation.

FIGS. 3A-E demonstrate Acacia mangium regeneration from stem pieces.FIG. 3A shows regeneration from callus. FIG. 3B shows adventitious budsinduced from callus. FIG. 3C show root formation, FIG. 3D shows acomplete plantlet and FIG. 3E shows a one month old plantlet in potsoil.

FIG. 4 is a map of pBI121 with an 800 bp HindIII-BamHI fragmentcontaining the cauliflower mosaic virus (CaMV) 35S promoter clonedupstream of the GUS gene.

FIGS. 5A-C demonstrate rejuvenation of a tree. FIG. 5A shows a two-yearold tree. FIG. 5B shows adventitious bud induction. FIG. 5C showspropagated adventitious buds with plumules.

FIG. 6A shows adventitious buds and FIG. 6B shows stem pieces asexplants for transformation.

FIG. 7 demonstrates selection and induction of putative transgenicadventitious buds.

FIGS. 8A and 8B illustrate GUS staining of adventitious buds afterselection for 5 months.

FIGS. 9A-C illustrate GUS staining of young transgenic stem pieces.FIGS. 9A-B show stem pieces and FIG. 9C shows a shoot.

FIGS. 10A-C show GUS staining of transgenic leaf and leaf pieces.

FIG. 11 shows the results of a Southern blot to a NPTII probe. 20 μg ofDNA was digested with HindIII, run on a gel and blotted. Lanes 1-6 aretransgenic lines. Lane ck is a negative control using DNA from anon-transgenic plant. Lane 7 is a positive control using DNA from atomato transgenic line by plasmid pWS42 with NPTII as the selectionmarker.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for geneticallytransforming tissue of trees, especially Acacia mangium, andregenerating whole plants via organogenesis from the transformed tissue.

The present invention is further detailed in the following Examples,which are offered by way of illustration and are not intended to limitthe invention in any manner. Standard techniques well known in the artor the techniques specifically described below are utilized.

EXAMPLE 1

Reagents and Culture Media

A. MS Basic Medium: MS Medium (Murashige and Skoog, 1962) SigmaConcentration Cat. Name Molecular formula (mg/l) No. MacronutrientsAmmonium Nitrate NH₄NO₃ 1,650 A-3795 Potassium Nitrate KNO₃ 1,900 P-8291Calcium Chloride CaCl₂.2H₂O 440 C-2536 dihydrate Magnesium SulfateMgSO₄.7H₂O 370 —7774 Heptahydrate Potassium phosphate KH₂PO₄ 170 P-8416monobasic, anhydrous Ferrous Sulfate FeSO₄.7H₂O 27.8 F-8263 HeptahydrateEthylenediamine- C₁₀H₁₄N₂O₈Na₂.2H₂O 37.3 E-6635 tetraacetic acid (EDTA)(Na₂EDTA) Micronutrients Potassium Iodide KI 0.83 P-8166 Boric AcidH₃BO₃ 6.2 B-9645 Manganese Sulfate MnSO₄.H₂O 16.9 —7899 monohydrate ZincSulfate ZnSO₄.7H₂O 8.6 Z-1001 Molybdic Acid Na₂MoO₄.2H₂O 0.25 —1651(Sodium salt:dihydrate) Cupric Sulfate CuSO₄.5H₂O 0.025 C-3036(Pentahydrate) Cobalt Chloride CoCl₂.6H₂O 0.025 C-2911 (hexahydrate)Organic Reagents myo-Inositol C₆H₁₂O₆ 100 I-3011 Nicotinic acid C₆H₅NO₂0.5 —0765 Glycine C₂H₅NO₂ 2.0 G-6143 Thiamine C₁₂H₁₇ClN₄OS.HCl 0.1T-3902 (Vitamin B1) Pyridoxine C₈H₁₁NO₃.HCl 0.5 P-9755 (Vitamin B6)Hydrochloride

B. Other Organic Reagents Sigma Cat. Name Molecular formula No.L-Ascorbic acid (Vitamin C) C₆H₈O₆ A-2174 Casein Enzymatic Hydrolysate(CH) C-7290 L-Glutamine (Gln) C₅H₁₀N₂O₃ G-9273 L-Asparagine monohydrate(Asn) C₄H₈N₂O₃.H₂O A-4284 L-Proline (Pro) C₅H₉NO₂ P-4655 Sucrose S-5390

C. Plant Growth Regulators Sigma Cat. Name No. Indole-3-acetic Acid(IAA) I-2886 α-Naphthaleneacetic Acid(NAA) —06401-Phenyl-3-(1,2,3-thiadiazol-5-yl)Urea (Thidiazuron, TDZ) P-61866-Benzylaminopurine (6-BA) B-3408 Kinetin (KT) K-0753 Gibberellic Acid(GA₃) G-7645

D. Agar Sigma Cat. Name No. Type M A-4800 Purified A-7921 PhytagelP-8169 E. Antibiotics Timentin (T) Beecham Pharmaceuticals (Pte) LtdKanamycin (K) Sigma Genetins (G418 Sulfate) Clontech 8056-2

F. Media for Callus Induction AM-5 MS + 2,4-D 2.0 mg/l + KT 3.0 mg/lAM-6 MS + 2,4-D 2.0 mg/l + KT 0.5 mg/l AM-7 MS + NAA 2.0 mg/l + KT 3.0mg/l AM-8 MS + NAA 2.0 mg/l + KT 0.5 mg/l AM-14 MS + 2,4-D 0.5 mg/l +6-BA 3.0 mg/l AM-15 MS + 2,4-D 1.0 mg/l + 6-BA 3.0 mg/l AM-16 MS + 2,4-D2.0 mg/l + 6-BA 3.0 mg/l AM-17 MS + 2,4-D 0.5 mg/l + 6-BA 0.5 mg/l AM-18MS + 2,4-D 1.0 mg/l + 6-BA 0.5 mg/l AM-19 MS + 2,4-D 2.0 mg/l + 6-BA 0.5mg/l AM-20 MS + 2,4-D 0.5 mg/l + 6-BA 1.0 mg/l AM-21 MS + 2,4-D 1.0mg/l + 6-BA 1.0 mg/l AM-22 MS + 2,4-D 2.0 mg/l + 6-BA 1.0 mg/l AM-27MS + NAA 0.5 mg/l + 6-BA 3.0 mg/l AM-28 MS + NAA 1.0 mg/l + 6-BA 3.0mg/l AM-29 MS + NAA 2.0 mg/l + 6-BA 3.0 mg/l AM-30 MS + NAA 0.5 mg/l +6-BA 0.5 mg/l AM-31 MS + NAA 1.0 mg/l + 6-BA 0.5 mg/l AM-32 MS + NAA 2.0mg/l + 6-BA 0.5 mg/l AM-33 MS + NAA 0.5 mg/l + 6-BA 1.0 mg/l AM-34 MS +NAA 1.0 mg/l + 6-BA 1.0 mg/l AM-35 MS + NAA 2.0 mg/l + 6-BA 1.0 mg/lAM-231 MS + 2,4-D 5.0 mg/l + KT 0.5 mg/l AM-233 MS + 2,4-D 0.5 mg/l + KT1.0 mg/l AM-234 MS + 2,4-D 1.0 mg/l + KT 1.0 mg/lG. Media for adventitious buds induction

-   a. AM-265: MS basic medium with TDZ 1.0 mg/l, IAA 0.25 mg/l, CH 100    mg/l, Vc 100 mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH    5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8%    (A-4800, Sigma), sucrose 30 g/L.-   b. AM-261: MS basic medium with TDZ 1.0 mg/l, IAA 0.5 mg/l, CH 100    mg/l, Vc 100 mg/l, Gln 150 mg/l, Asn 150 mg/l, Pro 150 mg/l, pH 5.8    after autoclave, phytagel 0.275 or 0.30%, or agar 0.8% (A-4800,    Sigma), sucrose 30 g/L.-   c. AM-304: MS basic medium with TDZ 2.0 mg/l, IAA 0.25 mg/l, CH 100    mg/l, Vc 100 mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH    5.8 after autoclave, phytagel 0.275 or 0.30%, or agar 0.8% (A-4800,    Sigma), sucrose 30 g/L.-   d. AM-308: MS basic medium with TDZ 1.0 mg/l, IAA 2.0 mg/l, CH 100    mg/l, Vc 100 mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH    5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8%    (A-4800, Sigma), sucrose 30 g/L.    H. Media for Pinnate Leaf Formation-   a. AM-337: MS basic medium with TDZ 0.01 mg/l, CH 100 mg/l, Vc 100    mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH 5.8 after    autoclaving, phytagel 0.275 or 0.30%, or agar 0.8% (A-4800, Sigma),    sucrose 30 g/L.-   b. AM-4 1: MS basic medium supplemented with 6-BA 2 mg/l, CH 100    mg/l, Vc 100 mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH    5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8%,    sucrose 30 g/L.    I. Media for Root Formation-   a. Modified AM-8: MS basic medium with NAA 2.0 mg/l, KT 0.5 mg/l,    CH1100 mg/l, Vc 100 mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150    mg/l, pH 5.8 after autoclaving, phytagel 0.30%, sucrose 30 g/L.-   b. AM-357: /2 MS basic medium with NAA 2.0 mg/l, KT 0.5 mg/l, CH 100    mg/l, Vc 100mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH    5.8 after autoclaving, phytagel 0.30%, sucrose 30 g/L.-   c. AM-45 1: V₂MS basic medium with NAA 2.0 mg/l, KT 0.1 mg/l,CH 100    mg/l, Vc 100 mg/l, Gln 150 mg/l, Asn 150 mg/l and Pro 150 mg/l, pH    5.8 after autoclaving, phytagel 0.35%, sucrose 20 g/L.    J. Transplantation

Peat soil: white sand 3:1

K. Media for Agrobacterium tumefaciens Activation LB medium Tryptone 1.0% Yeast extract 0.5 % NaCl 0.8 % pH 7.0 before autoclaving (Sambrook etal., 1989) YEP medium (Per liter) (Chilton et al., 1974) Bactopeptone 10g Yeast extract 10 g NaCl 5 g AB medium 20 × Phosphate Buffer (perliter) K₂HPO₄ 60 g NaH₂PO₄ 20 g Autoclave this solution separately 20 ×Salts solution (per liter) NH₄Cl 20 g MgSO₄.7H₂O 6 g KCl 3 g CaCl₂ 0.2 gFeSO₄.7H₂O 0.05 g pH to 7.0 before autoclaving To make up the finalmedium, combine (to a final volume of 1 liter): 50 ml 20 × PhosphateBuffer 50 ml 20 × Salts Solution 900 ml sterile ddH₂0 Induction mediumMES buffer, pH 6.0 30 mM 1 × AB medium Glucose 0.5 % Acetosyringone 100μM (a stock solution of acetosyringone must be made up fresh in DMSO)

EXAMPLE 2

Assay for GUS Activity

Stain the tissue overnight at 37° C. in GUS staining solution. Gusstaining solution is described by Jefferson (1987). It is X-gluc—1 mM,sodium phosphate (pH 7.0)—100 mM, EDTA—10 mM, and Triton X-100—0.1%. GUSstaining showed a positive blue reaction in adventitious buds and stemand leaf (FIGS. 8, 9, and 10).

EXAMPLE 3

Southern Analysis

A method for performing Southern blots is described in Sambrook et al.(1989). The method is:

A. DNA Extraction

2-5 g fresh sample was frozen in liquid N₂. This was ground with amortar and pestle in liquid nitrogen to a fine powder. The powder wastransferred to a centrifuge tube (50 ml). 15 ml of Extraction Buffer wasadded, 2 ml 10% SDS was added and mixed thoroughly. This was incubatedat 65° C. for 15 minutes. 5 ml 5 M KAc was added and shaken vigorously.The mixture was incubated in ice for 20 minutes and then spun at25,000×g for 20 minutes. The supernatant was filtered through Microclothinto a new tube. The DNA was precipitated with ½ volume of isopropanol,mixed and incubated at −20° C. for 30 minutes.

The DNA was pelleted at 25,000×g for 30 minutes, the supernatant waspoured off, and the tube was inverted and air dried for 30 minutes. Thepellet was dissolved with 0.7 ml of 1× TE (pH 8.0) and transferred to anEppendorf tube. This was spun 10 minutes. The supernatant wastransferred to a new tube, 7 μl RNase (10 mg/ml) was added and left atroom temperature for 10 minutes, then 75 μl 3 M NaAc (pH 5.3) and 500 μlof isopropanol were added. The solution was mixed and the DNA waspelleted in a microcentrifuge at full speed for 5 minutes. The pelletwas washed with 70% ethanol, air dried and dissolved with 100 μl 1× TE(pH 8.0).

B. Extraction Buffer 100 mM Tris-HCl, pH 8 50 ml  50 mM EDTA, pH 8 50 ml500 mM NaCl 50 ml  10 mM β-ME 0.6 ml ddH₂O to 500 mlC. Enzyme Digestion

Reaction system I: DNA sample 100 μl (20 μg), 10× HindIII buffer 40 μl,HindIII 8 μl (80 units), add sterile double distilled H₂O 252 μl tototal volume 400 μl.

The reaction was incubated at 37° C. for overnight. 40 μl of 3 M NaAc(pH 5.3) and ⅔ volume of 100% ethanol were added to the reaction systemand this was incubated at −20° C. for 30 minutes. This was spun at fullspeed at 4° C. for 20 minutes. The supernatant was poured off and thetube was air dried for 30 minutes then the pellet was dissolved in 30μll sterile double distilled water.

D. Electrophoresis

Electrophoresis was performed on a 0.8% agarose gel in 1×TBE at 28V forovernight.

E. Transfer of DNA from Agarose Gels to Nylon Membranes

This step was performed as described in Sambrook et al. (1989).

F. Prehybridization

A prehybridization solution of 6×SSC, 5× Denhardt's reagent, 0.5% SDS,100 μg/ml denatured, fragmented salmon sperm DNA (Stratagene products)and 50% formamide was prepared. 50× Denhardt's is: 5 g of Ficoll, 5 g ofpolyvinylpyrrolidone, 5. g of bovine serum albumin and ddH₂O to 500 ml,filtered and stored at −20° C.

After fixing DNA to the membrane, the membrane was placed into ahybridization tube containing suitable prehybridization solution using0.2 ml prehybridization solution for each square centimeter of nylonmembrane. The membrane was incubated at 42° C. for 6 hours.

G. Probe Label

During the prehybridization, labeled probe was prepared using aBoehringer Mannheim-High Primer DNA Labeling Kit. 50 ng of NPTII wereadded then brought to a volume of 8 μl with H₂O. The DNA was denaturedin a 100° C. heat block for 10 minutes, chilled quickly in ice, andpulse spun.

On ice the denatured DNA was mixed with: 4 μl High Prime reactionmixture, 3 μl of dATP, dGTP, dTTP mixture, and 5 μl of CC-³²P dCTP, 3000Ci/mmol (Biolab). This was incubated at 37° C. for 10 minutes. Thereaction was stopped by adding 20 μl of 50 mM EDTA (pH 8.0).

The labeled probe was purified by running through a small Sephadex G50column prepared on a small Pasteur pipette. The eluent was monitoredwith a counter and the first peak was collected.

H. Hybridization

The probe was added into the hybridization tube, then incubated at 42°C. for 10-24 hours.

I. Washing Membrane

The hybridization solution was poured off and the membrane was submergedin 2×SSC, 0.5% SDS at room temperature for 10 minutes. The membrane wastransferred into 2×SSC, 0.1% SDS at room temperature for 15 minutes. Thesolution was replaced with 0.5×SSC, 0.1% SDS and the membrane wasincubated at room temperature for 15 minutes. The solution was replacedwith 0.1×SSC, 0.1% SDS and incubation continued at 55° C. for 30-55minutes. The membrane was transferred into 0.1 ×SSC at room temperaturefor 3-5 minutes, then air dried on 3MM Whatman paper for 30 minutes.

J. Autoradiography

The membrane was exposed to X-ray film (Kodak) to obtain anautoradiographic image at −80° C. for one day or more.

Using NPTII fragment as a probe, Southern blotting showed that the NPTIIgene had integrated in adventitious buds (FIG. 11). The resultsdemonstrate that this protocol of Acacia mangium transformation is verysuccessful.

EXAMPLE 4

Regeneration of A. mangium

A. Embryo Culture

Mature seeds (black coat) were pre-treated with 98% H₂SO₄for 2-3 minutesand washed with tap water several times. Treated seeds were sterilizedwith 70% ethanol for 2-3 minutes and washed times with sterile ddH₂O.Seeds were then immersed in 0.1% HgCl₂ for 6 minutes and washed 5 timeswith sterile ddH₂O, again sterilized with bleach 30% (market product)for 6 minutes, then washed 5 times with sterile ddH₂O. Sterilized seedswere soaked in sterile ddH₂O overnight for isolating zygotes for embryoculture. MS basic medium (Murashige and Skoog, 1962), pH 5.8, sucrose 30g/L, phytagel 0.25% or agar 0.7% (Sigma, A-4800), with or withoutactivated charcoal, was used to culture mature embryo. Isolated maturezygotic embryos were cultured on MS using a photoperiod of 12/12 hoursor 16/8 hours (L/D) or complete dark, at 25-28° C. Germinated hypocotylsor leaves or petioles or stems were used as explants to induce callusformation.

B. Callus Induction

Different media were used to induce callus, including medium Nos. AM-5,6, 7, 8, 14, 15, 16, 17, 18, 19, 20, 21, 22, 27, 28, 29, 30, 31, 32, 33,34, 35, 231, 233 and 234 at 12/12 hours or 16/8 hours (L/D) or Dark at25° C. Callus induced from young leaflet: AM-L; from young petiole:AM-P; from hypocotyl: AM-H; from young stem: AM-S; from bud: AM-B; fromroot: AM-R. All above calli were used to induce adventitious buds, usinga photoperiod of 16/8 hours, 1800-2000 lux, and 28° C. The results ofcallus induction on different media were different (Table. 1). Generallycallus induction was not difficult. All explants produced calli at thephotoperiod of 12/12 hrs or 16/8 hrs (L/D) (see, e.g., FIG. 1A; FIG. 2A;FIG. 3A), and 25° C. for 20 days. TABLE 1 Results of callus induction ondifferent media at 12/12 hours (L/D), 25° C. cultured for 35 days No. ofexplants Number of No. of explants complete callus Medium No. Explants(leaflet) forming callus formation AM-5 40 40 33 AM-6 40 40 31 AM-7 4040 3 AM-8 40 40 4 AM-14 40 40 36 AM-15 40 40 40 AM-16 40 40 38 AM-17 4040 36 AM-18 40 40 39 AM-19 40 40 39 AM-20 40 40 38 AM-21 40 40 40 AM-2240 40 40 AM-27 40 40 12 AM-28 40 40 8 AM-29 40 40 11 AM-30 40 40 10AM-31 40 40 6 AM-32 40 40 2 AM-33 40 40 4 AM-34 40 40 4 AM-35 40 40 6AM-231 40 40 30 AM-233 40 40 20 AM-234 40 40 20 Petiole AM-6 20 20 15AM-17 20 20 20C. Adventitious Bud Induction

Calli induced from leaflet, petiole, young stem and buds were culturedon AM-261, AM-265, AM-304, and AM-308, at a photoperiod of 16/8 hrs(L/D), at 28° C. One month later, there were some bud recoveries fromcallus (FIG. 1B; FIG. 2B). Bud recovery from friable callus firstappeared as compact and smooth bud terminal. The ratio of bud recoveryreaches 15% on AM-265. But on these media, induced buds could not easilyform pinnate leaves and elongate.

D. Pinnate Leaf Formation and Adventitious Bud Elongation

Bud recoveries from callus were transferred into AM-337 or AM-41 withGA₃ 2.5 mg/l, at a photoperiod of 16/8 hrs (L/D), 1800-2000 lux, and 28°C. After culture for one month, some of them could form pinnate leavesand elongate (FIGS. 1C, D, E; FIG. 2C; and FIG. 3B). The ratio ofpinnate leaf formation and bud elongation reaches 16.67%.

E. Root Formation

Elongated buds were cultured in AM-357 or AM-451 for root formation. 20days later, adventitious roots began to appear from basic stem of buds(FIG. 3C). After root formation, plantlets were transferred to MS basicmedium without any plant growth regulators. Plantlets grow normally andfast, and the root system grew well (FIG. 3D).

F. Transplantation

Phytagel on plantlets was washed away and the plantlets were transferredto pot (peat soil: white sand 3:1) and grown in a growth chamber at aphotoperiod of 16/8 hours and 25° C. One month later the plantlets weretransferred to a green house (FIG. 3E).

EXAMPLE 5

Rejuvenation of Tree

The auxiliary buds of a two-year old young tree (FIG. 5A) were culturedon MS basic medium with NAA 0.1 mg/l, 6-BA 3.0 mg/l, CH1100 mg/l, Vc 100mg/l, Pro 150 mg/l, Asn 150 mg/l and Gln 150 mg/l, phytagel 0.275% oragar 0.8% (A-4800, Sigma), pH 5.8 after autoclaving at 121° C., sucrose30 g/L. Sixty days later, some adventitious buds with phyllodes wereobtained (FIG. 5B). Induced adventitious buds with phyllodes weresubcultured on AM-41 (MS basic medium with 6-BA 2 mg/l, CH 100 mg/l, Vc100 mg/l, Pro 150 mg/l, Asn 150 mg/l and Gln 150 mg/l, phytagel 0.275%or agar 0.8% (A-4800, Sigma), pH 5.8 after autoclaving at 121° C.,sucrose 30 g/L). After sub-culturing adventitious buds with phyllodestwice in about two months, the rejuvenation of adventitious shoots withpinnate leaf was obtained (FIG. 5C). Adventitious buds can be used asexplants for transformation (FIG. 6A).

EXAMPLE 6

A. mangium Genetic Transformation

A. Activation of Agrobacterium tumefaciens Strain LBA4404/pB121

Agrobacterium strain LBA4404 (Ooms et al., 1981) harbouring binaryvector plasmid pBI121 (size of vector 13.0 kb, Clontech, FIG. 4) wasused for this experiment. pB 121 was derived from pBI101 with an 800-bpHindIII-BamHI fragment containing the cauliflower mosaic virus (CaMV)35S promoter cloned upstream of the GUS gene. From a permanent glycerolstock stored at −70° C., Agrobacterium tumefaciens pBI12I/LBA4404 wasstreaked onto a solid LB (pH 7.0) medium plate containing Streptomycin100 mg/l (Str¹⁰⁰) and Kanamycin 50 mg/l (K⁵⁰). This was incubated 2-3days at 28° C. Fresh pBI121/LBA4404 was streaked onto solid LB medium(pH 7.0) with Streptomycin 100 mg/l (Str¹⁰⁰) and Kanamycin 50 mg/l (K⁵⁰)for overnight or 24 hours at 28° C. the dark. Several colonies ofpBI121/LBA4404 were picked and pBI121/LBA4404 was inoculated in liquid50 ml LB medium with Streptomycin 100 mg/l (Str¹⁰⁰) and Kanamycin 50mg/l (K⁵⁰), and cultured at 28° C. under dark, 250 rpm for 10 hoursuntil OD₆₀₀=0.70-1.10. This was spun down at 3500 rpm for 30 minutes or5000 rpm for 10 minutes, and resuspended in 4-5 volumes of YEP medium(pH 7.0) with Streptomycin 100 mg/l (Str¹⁰⁰) and Kanamycin 50 mg/l(K⁵⁰), OD₆₀₀=0.10-0.20, incubated at 28° C. in the dark, 250 rpm for8-10 hours, OD₆₀₀=0.70-1.20. This at 3500 rpm for 30 minutes andresuspended in the same volume of sterile NaC10.9%. This was spun downat 3500 rpm for 30 minutes and resuspended in 2-3.5 volumes of inductionmedium, OD₆₀₀=0.2-0.3, and incubated at 28° C. in the dark, 120 rpm for8-15 hours, OD₆₀₀=0.70-1.20 for infecting explants or callus or a cellsuspension.

B. Preculture of Stem Pieces

Stem pieces were cultured on AM-265 for 3 days using a photoperiod of16/8 hrs (L/D), 1800-2000 lux, at 28° C.

C. Co-cultivation

After preculture, stem pieces were soaked in 0.5 M mannitol for 20-25minutes and then transferred to activated pBI121/LBA4404 suspension for15 minutes. Infected young stem pieces were washed once and dried onsterile Whatman paper before being cultured on AM-265 with 100 μMacetosyringone at pH 5.2 at 22° C. in the dark for 3 days.

D. Selection of Transformed Adventitious Buds and Transformed Callus

After co-culturing for 3 days, stem pieces were washed with sterileddH₂O for 10 times and dried on sterile Whatman paper. Then stem pieceswere cultured on AM-265 containing Timentin 250 mg/l and G418 12 mg/l,phytagel 0.275%, using a photoperiod of 16/8 hrs (L/D) and 28° C. forselecting transformed callus or transformed adventitious buds.Subsequently, stem pieces were cultured on the above media with G418 12mg/l for 25 days, G418 20 mg/l for 60 days, G418 30 mg/l for 25 days,then on G418 12 mg/l. After continuous selecting for 4 months onantibiotics, 33.75% of stem pieces formed adventitious buds, and 2.5mg/l to 5 mg/l GA₃ was added into the same media to promote adventitiousbud elongation (FIG. 7). After selection for five months, Timentin wasno longer used in the medium and some adventitious buds were used forGUS staining. GUS staining showed positive blue colour reaction inadventitious buds (FIG. 8, FIG. 9, and FIG. 10). Adventitious buds weretransferred to AM-41 with GA₃ 2.5 mg/l to promote pinnate leafformation.

E. Root Formation

Transformed adventitious buds were transferred into AM-357 or AM-451with or without G418 (10 mg/l) using a photoperiod of 16/8 hrs (L/D) at28° C.

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

List of References

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1. A method for regenerating Acacia mangium comprising: a) inducingcallus formation from an explant; b) culturing the callus on a mediumcomprising thidiazuron to produce adventitious buds; c) culturing theadventitious buds to elongate the buds and produce pinnate leaves; andd) culturing elongated buds of step (c) to produce plantlets with roots.2. The method of claim 1 wherein seeds are cultured to produce theexplant.
 3. The method of claim 1 wherein the explant is selected fromthe group consisting of hypocotyls, cotyledons, leaves, petioles andstems.
 4. The method of claim 1 wherein the explant is cultured on amedium comprising MS medium supplemented with an auxin and a cytokinin.5. The method of claim 4 wherein the auxin is present at 0.5-2.0 mg/land the cytokinin is present at 0.5-3.0 mg/l.
 6. The method of claim 4wherein the auxin is selected from the group consisting of 2,4-D andα-naphthaleneacetic acid and wherein the cytokinin is selected from thegroup consisting of kinetin and 6-benzylaminopurine.
 7. The method ofclaim 1 wherein the callus is cultured on a medium comprising MS basicmedium supplemented with a) thidiazuron, b) indole acetic acid, c)casein enzymatic hydrolysate, d) L-ascorbic acid, e) L-glutamine, f)L-asparagine, g) L-proline, h)sucrose and i) agar or phytagel.
 8. Themethod of claim 1 wherein the adventitious buds are cultured on a mediumcomprising MS medium supplemented with a) thidiazuron, b) caseinenzymatic hydrolysate, c) L-ascorbic acid, d) L-glutamine, e)L-asparagine, f) L-proline, g) sucrose and h) agar or phytagel.
 9. Themethod of claim 1 wherein the elongated buds are cultured on a mediumcomprising ½ MS basic medium supplemented with a) α-naphthaleneaceticacid, b) kinetin, c) casein enzymatic hydrolysate, d) L-ascorbic acid,e) L-glutamine, f) L-asparagine, g) L-proline, h) sucrose and i)phytagel.
 10. The method of claim 1 wherein the explant is transformed.11. A method for regenerating Acacia mangium comprising: a) culturingauxiliary buds from an Acacia mangium tree to produce adventitious budscomprising phyllodes; b) subculturing the adventitious buds comprisingphyllodes to produce adventitious shoots; c) culturing the adventitiousshoots.
 12. The method of claim 11 wherein the culturing of auxiliarybuds is performed on a medium comprising MS basic medium supplementedwith a) α-naphthaleneacetic acid, b) 6-benzylaminopurine, c) caseinenzymatic hydrolysate, d) L-ascorbic acid, e) L-proline, f)L-asparagine, g) L-glutamine, h) sucrose, and i) phytagel or agar. 13.The method of claim 11 wherein the subculturing of adventitious budscomprising phyllodes is performed on a medium comprising MS basic mediumsupplemented with a) 6-benzylaminopurine, b) casein enzymatichydrolysate, c) L-ascorbic acid, d) L-glutamine, e) L-asparagine, f)L-proline, g) sucrose and h) phytagel or agar.
 14. A method of makingtransgenic Acacia mangium plants comprising; a) preparingtransgenicAcacia mangium cells by preculturing stem pieces in culturemedium comprising thidiazuron; b) co-cultivating the stem pieces of step(a) with Agrobacterium in medium comprising thidiazuron to formadventitious buds; c) culturing the adventitious buds in a selectivemedium comprising an antibiotic and gibberellic acid; and d) rooting theplantlets which develop from the adventitious buds of step c).
 15. Themethod of claim 14 wherein the selective medium comprises G418.
 16. Themethod of claim 15 wherein the adventitious buds are cultured for morethan 1 month.
 17. A transgenic Acacia mangium plant.
 18. A transgeniccell of the Acacia mangium plant of claim 17.